WO2008091930A2 - Marqueurs rapporteurs pouvant être scindés et procédés d'utilisation - Google Patents

Marqueurs rapporteurs pouvant être scindés et procédés d'utilisation Download PDF

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Publication number
WO2008091930A2
WO2008091930A2 PCT/US2008/051773 US2008051773W WO2008091930A2 WO 2008091930 A2 WO2008091930 A2 WO 2008091930A2 US 2008051773 W US2008051773 W US 2008051773W WO 2008091930 A2 WO2008091930 A2 WO 2008091930A2
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WIPO (PCT)
Prior art keywords
reporter
reporter tag
tag
scissile
tags
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WO2008091930A3 (fr
Inventor
Michelle D. Guerra
Edward Papacoda
Rixin Wang
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Revvity Health Sciences Inc
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PerkinElmer LAS Inc
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Publication of WO2008091930A2 publication Critical patent/WO2008091930A2/fr
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Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • Certain examples of the technology described herein are directed to scissile reporter tags and methods of using them. More particularly, in certain embodiments, an isobaric tag comprising at least two scissile bonds is provided.
  • a set comprising a first reporter tag and a second reporter tag.
  • the first reporter tag comprises at least two scissile bonds
  • the second reporter tag comprises at least two scissile bonds.
  • the first reporter tag is constructed and arranged to be distinguished from the second reporter tag after cleavage of each of the first reporter tag and the second reporter tag at one or more of the at least two scissile bonds.
  • the at least two scissile bonds of the first reporter tag have a spacing between them that is different than a spacing between the at least two scissile bonds of the second reporter tag.
  • the spacing between the at least two scissile bonds of the first reporter tag is six amino acid residues and the spacing between the at least two scissile bonds of the second reporter tag is seven amino acid residues.
  • the first reporter tag and the second reporter tag comprise a common property.
  • the common property is selected from the group consisting of mass, mass-to- charge ratio, composition and at least one heavy isotope.
  • the first reporter tag and the second reporter tag comprise a different distribution of heavy isotopes.
  • each of the first reporter tag and the second reporter tag are configured as a peptide reporter tag.
  • each of the first reporter tag and the second reporter tag comprises at least one moiety for attachment to a peptide.
  • the scissile bond of each of the first reporter tag and the second reporter tag is a peptide bond between an A-B sequence, wherein B is proline and A is selected from the group consisting of alanine, glutamic acid (or glutamate), phenylalanine, isoleucine, serine, aspartic acid (or aspartate), and asparagine.
  • At least one of the first and second scissile bonds of each of the first reporter tag and the second reporter tag is a peptide bond between an aspartic acid-proline sequence. In some examples, each of the first and second scissile bonds of each of the first reporter tag and the second reporter tag is a peptide bond between an aspartic acid- proline sequence. In certain examples, at least one of the first and second scissile bonds of each of the first reporter tag and the second reporter tag is a peptide bond between an A-B sequence, in which A is asparagine and B is selected from the group consisting of leucine, threonine, and serine.
  • the set may further comprise at least one additional reporter tag including at least two scissile bonds.
  • the at least one additional reporter tag may be constructed and arranged to be distinguished from the first reporter tag and the second reporter tag after cleavage of each of the first reporter tag, the second reporter tag, and the additional reporter tag at one or more of the at least two scissile bonds.
  • the set of reporter tags comprises at least six different reporter tags, at least ten different reporter tags or at least fifteen different reporter tags.
  • a set of reporter tags comprises at least a first reporter tag and a second reporter tag.
  • each of the first and second reporter tags comprises a first scissile bond and a second scissile bond.
  • SUBSTITUTE SHEET bonds of the first reporter tag is different than a spacing between the first and second scissile bonds of the second reporter tag to distinguish the first and second reporter tags from each other.
  • the different spacing of the first and second scissile bonds of the first and second reporter tags provides at least one distinguishable fragment after cleavage at one scissile bond in each of the first and second reporter tags.
  • the spacing between the first and second scissile bonds of the each of the first and second reporter tags is between six and ten amino acid residues provided that the spacing between the first and second scissile bonds of the first reporter group is different than the spacing between the first and second scissile bonds of the second reporter group.
  • the first and second reporter tags are each configured as isobaric tags or peptide reporter tags.
  • the set further comprises a third reporter tag.
  • the third reporter tag comprises a first scissile bond and a second scissile bond having a spacing from the first scissile bond.
  • the spacing between the second scissile bond and the first scissile bond of the third reporter tag may be selected to distinguish the first, second and third reporter tags from each other.
  • the spacing between the first and second scissile bonds of the first reporter tag is six amino acid residues
  • the spacing between the first and second scissile bonds of the second reporter tag is seven amino acid residues
  • the spacing between the first and second scissile bonds of the third reporter tag is eight amino acid residues.
  • a set comprising a first peptide reporter tag and a second peptide reporter tag.
  • the first peptide reporter tag comprises at least two scissile bonds separated by a first quantity of amino acids.
  • the second peptide reporter tag comprises at least two scissile bonds separated by a second quantity of amino acids.
  • the first and second quantity of amino acids is different to distinguish the first peptide tag and the second peptide tag.
  • each of the first quantity of amino acids and the second quantity of amino acids is between six and ten provided that the first quantity of amino acids is different than the second quantity of amino acids.
  • the first and second peptide reporter tags are distinguishable after fragmentation in a mass spectrometer.
  • kits comprising at least two reporter tags.
  • the kit comprises a first reporter tag including at least two scissile bonds.
  • the kit further comprises a second reporter tag including at least two scissile bonds.
  • the first reporter tag is constructed and
  • the kit further comprises instructions for using the first and second reporter tags.
  • each of the first reporter tag and the second reporter tag may be configured as a peptide reporter tag.
  • the kit may further comprise at least one additional reporter tag including at least two scissile bonds.
  • the at least one additional reporter tag may be constructed and arranged to be distinguished from the first reporter tag and the second reporter tag after cleavage of each of the first reporter tag, the second reporter tag, and the additional reporter tag at one or more of the at least two scissile bonds.
  • a method comprising separating a first doubly-scissiled reporter tag coupled to a first target molecule and a second doubly-scissiled reporter tag coupled to a second target molecule.
  • the method may further comprise cleaving the first and second doubly-scissiled reporter tags.
  • the method may further comprise distinguishing the first doubly-scissiled reporter tag and the second doubly-scissiled reporter tag.
  • the method may further comprise configuring each of the first and second doubly-scissiled reporter tags to be distinguished from each other by varying a spacing between first and second scissile bonds in the first and second doubly-scissiled reporter tags.
  • the method may further comprise determining an amount of each of the first target molecule and the second target molecule. In other examples, the method may further comprise comparing the amount of the first target molecule and the amount of the second target molecule.
  • the method may further comprise distinguishing the first doubly-scissiled reporter tag and the second doubly-scissiled reporter tag by fragmenting the first doubly-scissiled reporter tag and the second doubly-scissiled reporter tag in a mass spectrometer and detecting at least one fragment from one of the fragmented, first doubly-scissiled reporter tag and the fragmented, second doubly-scissiled reporter tag.
  • the method may further comprise detecting at least one fragment from each of the fragmented first doubly-scissiled reporter tag and the fragmented second doubly-scissiled reporter tag.
  • a method of facilitating sequencing of a peptide comprises providing a set comprising a first reporter tag including at least two scissile bonds and a second reporter tag including at least two scissile bonds.
  • the first reporter tag may be constructed and
  • an isobaric reporter tag comprising at least two scissile bonds separated by a variable number of atoms to distinguish the isobaric reporter tag from a second isobaric reporter tag after cleavage of one or more of the at least two scissile bonds is disclosed.
  • the variable number of atoms separating the at least two scissile bonds comprises at least about 18 atoms.
  • the isobaric reporter tag may also include a linking group coupled to the isobaric reporter tag. In certain examples, the linking group may be coupled at a terminus of the isobaric reporter tag.
  • the linking group may be a haloacetyl or a pyridyl disulfide group.
  • the at least two scissile bonds are each a peptide bond between an aspartic acid residue and a proline residue and the linking group is an iodoacetyl group coupled to the aspartic acid residue of one of the at least two scissile bonds.
  • the iodoacetyl group may be coupled to an N-terminal aspartic acid residue.
  • FIG. 1 is a diagram showing cleavage of a reporter tag comprising two scissile bonds, in accordance with certain examples
  • FIG. 2 is another diagram showing cleavage of a reporter tag comprising two scissile bonds with one of the scissile bonds adjacent to a point of attachment to a target molecule, in accordance with certain examples;
  • FIG. 3 is schematic showing a set of peptide reporter tags and various fragments after cleavage of one or more scissile bonds, in accordance with certain examples.
  • FIG. 4 is a mass spectrum showing fragments from three peptide reporter tags, in accordance with certain examples.
  • compositions, kits, methods and the like disclosed herein provide a significant advancement in the ability to screen rapidly target molecules labeled with one or more of the reporter tags. Multiplex analysis may be performed using the compositions disclosed herein to identify a desired feature of a target molecule such as, for example, the amount of a target molecule present in a sample, the sequence of a target molecule, the presence or absence of a target molecule or other desired features selected by a user.
  • the reporter tags may be used individually or may be used in sets comprising two or more reporter tags. [0023] In accordance with certain examples, a set of reporter tags is provided.
  • reporter tag refers a composition that may be used to label, attach, link to or otherwise associate with a target molecule.
  • the reporter tag may also be used in conjunction with a target molecule without physical attachment or association.
  • the reporter tag may be configured as an isobaric label.
  • isobaric label or “mass tag” refers to a polymeric chain (e.g., of nucleic acids, peptides, peptide nucleic acids, etc.) of known mass that can attach to a target molecule, thereby adding its mass to the target molecule.
  • the reporter tag may be configured as a peptide reporter tag.
  • the reporter tag may be constructed and arranged such that it can be coupled to a target molecule.
  • the reporter tag may be constructed and arranged such that a target molecule coupled to a reporter tag can be distinguished from a target molecule not coupled to a reporter tag.
  • a target molecule coupled to a reporter tag may be separated from a target molecule not coupled to a reporter tag.
  • the reporter tag may be smaller in size and/or weight than the target molecule.
  • the term "couple” or “coupled” is used in the broad sense and is intended to encompass attachment or linkage by a covalent or a non-covalent bond to the target molecule.
  • the reporter tag 100 generally includes two or more scissile bonds, shown schematically as the bonds between groups A and B and groups C and D.
  • the term "scissile” refers to a bond that is labile or susceptible to breaking or cleavage at least under certain conditions, e.g., enzymatic treatment, collisional dissociation or the like. A scissile bond cleaves more easily
  • the labile or scissile bond is a photocleavable bond.
  • Photocleavable bonds are well known.
  • U.S. Patent No. 6,881,836 describes methods for synthesizing oligonucleotides and polypeptides containing one or more photocleavable bonds.
  • one or both of the labile scissile bonds in a reporter tag may be photocleavable.
  • the photocleavable bond may be broken, for example, by exposure to an ultraviolet (UV) light source of a MALDI laser.
  • UV ultraviolet
  • the labile or scissile bond is a peptide bond that is susceptible to breakage when subjected to collision induced dissociation (CID). In some embodiments, the labile or scissile bond is a peptide bond between an aspartic acid-proline (D-P) sequence.
  • CID collision induced dissociation
  • D-P aspartic acid-proline
  • fast atom bombardment collision-induced dissociation (FAB-CID) data indicates that the peptide bond between an A-B dipeptide sequence (where B is proline and A may be is alanine, glutamic acid (or glutamate), phenylalanine, isoleucine, or serine) fragments easily under the mass spectrometric conditions, but is relatively insensitive to the acid- or heat-mediated cleavage.
  • A-B dipeptide sequence where B is proline and A may be is alanine, glutamic acid (or glutamate), phenylalanine, isoleucine, or serine
  • one or both labile or scissile bonds in the reporter tag may be asparagine-proline (N-P). Peptides that contain this sequence undergo complete cleavage at the N-P amide bond after exposure to ammonia vapor or solution.
  • Other labile or scissile bonds include A-B bonds where A is asparagine and B is leucine (L), threonine (T), and serine (S). Asparagine residues not involved in chain-cleavage may undergo deamidation to aspartate upon exposure to ammonia.
  • one or both of the labile or scissile bonds of a reporter tag may be A-B bonds where A is phenylalanine, tyrosine or tryptophan and B may be any of the twenty commonly known amino acids.
  • one or both labile or scissile bonds of the reporter tag may include A-B bonds where A is arginine or lysine and B may be any of the twenty commonly known amino acids.
  • the scissile bond may be between an asparagine and a glycine residue may be cleaved in the presence of hydroxylamine at pH 9.
  • the scissile bond may be between asparagine and proline and may be cleaved in the presence of hydroxylamine at pH 2.5. Additional physical, chemical and enzymatic methods for cleaving scissile bonds will be readily selected by the person of ordinary skill in the art, given the benefit of this disclosure.
  • the bond between A and B may break, the bond between C and D may break or both bonds may break. Breaking or cleavage of the scissile bonds can provide multiple different fragments.
  • SUBSTITUTE SHEET example breaking of the scissile bond between groups A and B provides fragments 110 and 120. Breaking of the bond between groups C and D provides fragments 130 and 140. Breaking of both bonds provides fragments 150, 160 and 170.
  • the reporter tags disclosed herein are constructed and arranged such that at least one of fragments 110, 120, 130, 140, 150, 160 and 170 may be used to distinguish a first reporter tag from a second reporter tag.
  • the composition of the reporter tag may be selected such that at least one fragment is provided that is distinguishable from a fragment produced from other reporter tags.
  • the ability to distinguish multiple reporter tags in the same sample permits multiplex analysis, which may be used, for example, in protein sequencing operations or in analysis of multiple different samples simultaneously.
  • the spacing r between the groups that make up the scissile bonds may be variable.
  • r includes at least about 15 atoms between the B and C groups, more particularly, r includes at least about 18 atoms between the B and C groups.
  • the separation of the B and C groups may be up to about 24 atoms.
  • the reporter tag is configured as a peptide reporter tag
  • between about five and about ten amino acid residues are between the B and C groups, more particularly, about six to about nine amino acid residues are between the B and C groups, e.g., about six to about eight amino acids are between the B and C groups. It will be within the ability of the person of ordinary skill in the art, given the benefit of this disclosure, to select a desired spacing between the scissile bonds of the reporter tags disclosed herein.
  • the exact placement of the scissile bonds in the reporter tags may vary depending on the intended use of the reporter tag.
  • both scissile bonds are located within a backbone or chain of the reporter tag.
  • An example of this type of configuration is shown schematically in FIG. 1.
  • one or more of the scissile bonds may be located at a terminus or end of the reporter tag.
  • An example of this type of configuration is shown schematically in FIG. 2.
  • a reporter tag 200 is shown attached to a target molecule. The scissile bond between groups A and B or the scissile bond between groups C and D, or both, may be cleaved to provide various fragments.
  • fragment 210 is provided with the A group located at a terminus of the fragment that is attached to the target molecule.
  • Fragment 220 is also provided, which represents the remainder of the reporter tag.
  • the reporter tag may be a polymer comprising a plurality of monomers coupled to one another.
  • the reporter tag may be a comprised of amino acid monomers, thus forming a peptide reporter tag.
  • Another example is a reporter tag comprised of nucleic acid monomers, thus forming a nucleic acid reporter tag.
  • reporter tag that is a peptide nucleic acid reporter tag, comprised of monomeric synthetic homologs of nucleic acids that are coupled to one another along a pseudo-peptide backbone to form a PNA reporter tag (see, e.g., Paulasova and Pellestor, Ann. Genet. 47(4) 349-358, 2004).
  • the reporter tag may also be a hybrid, comprising more than one type of monomeric subunit (e.g., may be a mix of nucleic acid and amino acid monomeric subunits).
  • nucleic acid is intended to include ribonucleic acid, deoxyribonucleic acid, synthetic and natural derivates of these and nucleic acids that include bases other than adenine, thymine, guanine, cytosine and uracil.
  • the nucleic acid may include non-conventional nitrogenous bases such as, for example, hypoxanthine, xanthine, and uric acid. It will be within the ability of the person of ordinary skill in the art, given the benefit of this disclosure, to design suitable reporter tags for a desired use.
  • the reporter tag may be configured as a peptide reporter tag.
  • a peptide reporter tag is a tag that is used to label or attach to a target molecule and that includes at least one amino acid.
  • the peptide reporter tag may itself include a plurality of amino acids.
  • the peptide reporter tag may also include one or more linking groups to facilitate joining of the peptide reporter tag to a peptide or protein. Illustrative linking groups are discussed in more detail herein.
  • the groups that are between the two scissile bonds in the reporter tag may include one or more heavy isotopes to impart a different mass or a different mass-to-charge ratio to a particular fragment after cleavage of one or both of the scissile bonds of the reporter tag.
  • the heavy isotope may take different forms including deuterium, tritium, 13 C, 15 N, etc., or combinations thereof.
  • the reporter tag is configured as a peptide reporter tag
  • one or more amino acids containing an isotopically heavy nitrogen ( 15 N) and two isotopically heavy carbon atoms ( 13 C) may be used to provide a 3 Dalton shift.
  • glycine having a chemical formula of 13 C 2 H 3 15 NO may be used
  • SUBSTITUTE SHEET to provide a three (3) Dalton shift as compared to non-isotopically labeled glycine (C 2 H 3 NO).
  • C 2 H 3 NO non-isotopically labeled glycine
  • different fragments may be produced that have different masses (or mass-to-charge ratios).
  • mass-to-charge ratios By selecting two or more isobaric labels that provide fragments having close, but not the same, mass-to-charge ratios, the amount of each analyte present in a sample may be quantified by fitting of the peaks representative of the isobaric label fragments, as described for example in commonly owned U.S.
  • the target molecule may be an organic molecule or an inorganic molecule.
  • Illustrative types of target molecules include, but are not limited to, peptides, proteins, nucleosides, nucleic acids (e.g., DNA, RNA (such as mRNA, miRNA, or hnRNA)), fatty acids, carbohydrates, synthetic polymers (e.g., plastic or polyamide 6,6), metals and metals attached or coordinated to one or more complexes.
  • the reporter tags may be coupled to molecules being analyzed by mass spectrometry.
  • the mass spectrometry is tandem mass spectrometry, such as a MALD-QqTOF mass spectrometer, as discussed in more detail below.
  • a reporter tag may be coupled or directly associated with a target molecule.
  • a reporter tag may be coupled to an analyte by reactive groups on the target molecule, the reporter tag or both.
  • the reporter tag can be attached to analytes in numerous different manners.
  • a reporter tags may be covalently coupled to a protein through a disulfide bond between a cysteine group of the protein and a sulfhydryl group on the reporter tag.
  • coupling can be made using thiols, epoxides, nitrites for thiols, NHS esters, isothiocyanates for amines, and alcohols for carboxylic acids.
  • the reporter tag may include a suitable linking group or groups to attach or link the reporter tag to a target molecule of interest.
  • the linking group may include a leaving group that may be displaced during or after linking of the reporter tag to the target molecule. Suitable leaving groups include, but are not limited to, halides such as iodide and tosyl groups.
  • the reporter tag may be linked to the target molecule by formation of a covalent bond between the reporter tag and the target molecule. The formed covalent bond is preferably stronger than the scissile bonds discussed herein such that the target molecule-reporter tag product is not cleaved at the bond formed between them.
  • the reporter tag may be constructed and arranged such that attachment of the reporter tag to the target molecule occurs adjacent to one of the scissile bonds, as shown, for example, in FIG. 2.
  • the reporter tag is a peptide reporter tag
  • the first scissile bond preferably occurs between the first and second N-terminal amino acid residues of the peptide reporter tag.
  • the linking group of the reporter tag may be designed to provide selective reaction of the reporter tag with a desired group on the target molecule.
  • Illustrative linking groups that react with amines include, but are not limited to, imidoesters and N-hydroxysuccinimide (NHS) esters.
  • an imidoester comprising the reporter tag may react with an amine on the target molecule to provide an amidine linkage that attaches the reporter tag to the target molecule.
  • an NHS ester comprising the reporter tag may be formed and hydrolysis of the NHS ester, in the presence of a base, allows reaction between the reporter tag and an amino group of the target molecule.
  • particularly useful linking groups are those that selectively react with sulfhydryl groups on the target molecule.
  • Illustrative linking groups that react with sulfhydryls include, but are not limited to, haloacetyls and pyridyl disulfides.
  • a haloacetyl a haloacetyl compound comprising a reporter tag may be formed and reacted with a sulfhydryl containing target molecule to provide a haloacid and a target molecule linked to a reporter tag through an acetyl group.
  • a pyridyl disulfide compound comprising the reporter tag may be formed and reacted with a sulfhydryl containing target molecule. A disulfide exchange may occur such that the reporter tag is transferred to the target molecule and becomes attached through a disulfide bond.
  • hydrazides and carbodiimides may be used to attach the reporter tag to a target molecule containing an aldehyde and a carboxylic acid, respectively.
  • the reporter tag preferably includes only one primary amine, e.g., at the N-
  • SUBSTITUTE SHEET terminus in the case of a peptide reporter tag, to prevent any unwanted side reactions.
  • a carbodiimide such as EDC available from Pierce (Rockford, IL) may be reacted with a carboxyl group on the target molecule to form an o-acylisourea reactive ester (o-ARE).
  • o-ARE may react with a primary amine group of the reporter tag to form a target molecule linked to the reporter tag through an amide bond.
  • Illustrative linking groups suitable for use with the reporter tags disclosed herein are commercially available from Pierce (Rockford, IL).
  • reporter tags comprising amino acid, nucleic acid or both may be synthesized using conventional synthesis techniques.
  • nucleic acid reporter tags and peptide reporter tags may be synthesized using liquid phase synthesis, solid-phase synthesis, e.g., Merrifield synthesis, or other chemical techniques. See, e.g., Atherton, E., Sheppard, R.C. Solid Phase peptide synthesis: a practical approach. IRL Press, Oxford, England, 1989 and Stewart J.M., Young, J. D. Solid phase peptide synthesis, 2nd edition, Pierce Chemical Company, Rockford, 1984.
  • Nucleic acid reporter tags and peptide reporter tags may also be produced by inserting a suitable construct into a vector of a suitable biological system.
  • the reporter tag may take the form of a fusion construct, e.g., a GST or a His tag attached to the reporter tag, to facilitate isolation of the reporter tag.
  • Expression of the vector results in production of the reporter tag which may be subsequently isolated and purified using conventional purification techniques, e.g., column chromatography, immunoaffinity assays and the like. It will be within the ability of the person of ordinary skill in the art, given the benefit of this disclosure, to produce the reporter tags disclosed herein.
  • the reporter tags disclosed herein may be used in qualitative analysis. Without wishing to be bound by any particular scientific theory, qualitative analysis using the reporter tags disclosed herein typically involves assessing whether or not a particular fragment, or a target molecule attached to a fragment, is present in a sample or determining the sequence of a particular peptide or protein.
  • the reporter tags may be used to label substantially all peptides or proteins present in multiple samples, e.g., two, three or four samples.
  • the peptides or protein may be labeled before of after one or more treatment steps, e.g., enzymatic digestion with trypsin, chymotrypsin or the like.
  • the labeled samples may then be combined and subjected to analysis, i.e., multiplex analysis, by separating and analyzing the components in the sample in order to determine whether or not a particular fragment, and hence a particular labeled peptide, is present. Separation may be performed using many different methods such as for example, LC, HPLC,
  • a single target molecule from different samples may be labeled with different reporter tags.
  • the target molecule may be subjected to separation and/or analysis to determine the relative sizes of the labeled target molecules.
  • the target molecules may then be injected into a tandem mass spectrometer for analysis.
  • the labeled materials may be combined and fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
  • One or more regions of the polyacrylamide gel with at a desired molecular weight may be excised with a razor, and the gel piece macerated and subjected to trypsin digestion.
  • the resulting peptides may be desalted and concentrated using chromatography, e.g., high performance liquid chromatography, with a reverse-phase Cl 8 column and the purified peptides may be subjected to tandem mass spectrometry where the resulting peaks may be used to sequence the peptides.
  • the reporter tags may also be used in quantitative analysis. In a typical example, quantitative analysis may be used to assess the relative amounts of each target molecule present in a sample. An illustrative method for quantifying reporter tags and/or peptides attached thereto is described in more detail in U.S. 11/464,930 incorporated by reference above.
  • either the relative or absolute amount of a target molecule to which a reporter tag is attached may be determined.
  • quantitative analysis may be used to determine if a particular target molecule is present above a normal level, e.g., in a clinical diagnostic setting used to determine levels of various target molecules of clinical interest such as insulin, hormones, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), bilirubin, albumin, gamma-glutamyl transferase (GGT), lactic acid dehydrogenase (LDH), and other molecules commonly tested in blood analyses.
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • AST aspartate aminotransferase
  • bilirubin bilirubin
  • albumin gamma-glutamyl transferase
  • LDH lactic acid dehydrogenase
  • a single target molecule from different samples may be labeled with different reporter tags.
  • the target molecule may be subjected to separation and/or analysis to determine the relative amount of the labeled target molecules.
  • the target molecules may then be injected
  • the labeled materials may be combined and fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
  • One or more regions of the polyacrylamide gel with at a desired molecular weight may be excised with a razor, and the gel piece macerated and subjected to trypsin digestion.
  • the resulting peptides may be desalted and concentrated using chromatography, e.g., high performance liquid chromatography, with a reverse-phase Cl 8 column and the purified peptides may be subjected to tandem mass spectrometry where the resulting peaks may be used to sequence the peptides and/or determine the relative or absolute amounts of the peptides in the sample.
  • the reporter tags disclosed herein may be used or packaged in a set that includes two or more different reporter tags.
  • the first reporter tag of the set comprises at least two scissile bonds
  • the second reporter tag of the set comprises at least two scissile bonds.
  • the first reporter tag is constructed and arranged to be distinguished from the second reporter tag after cleavage of each of the first reporter tag and the second reporter tag at one or more of the at least two scissile bonds.
  • the first reporter tag and the second reporter tag may be distinguished after cleavage of both scissile bonds of each reporter tag.
  • distinguished refers to the case where the first and second reporter tags may be detected or identified in the presence of the other reporter tag. Distinguishing does not necessarily require baseline separation. Instead, as long as one fragment of a reporter tag may be identified or detected in the presence of another reporter tag (or fragment thereof), the reporter tags may be distinguished.
  • the at least two scissile bonds of the first reporter tag have a spacing between them that is different than a spacing between the at least two scissile bonds of the second reporter tag.
  • the number of atoms or residues that are between the scissile bonds of the first reporter tag may be different than the number of atoms or residues that are between the scissile bonds of the second reporter tag.
  • the difference in arrangement of the atoms or residues provides fragments having different mass or different mass-to-charge ratios, which permits distinction between the two reporter tags.
  • the spacing between the at least two scissile bonds of the first reporter tag may be six amino acid residues and the spacing between the at least two scissile bonds of the second reporter tag may be seven amino acid residues.
  • the first reporter tag and the second reporter tag comprise a common property.
  • the common property is selected from the group consisting of mass, mass-to-charge ratio, composition and at least one heavy isotope.
  • One particularly desirable common property is a similar mass-to-charge ratio, which allows the reporter tags to be used simultaneously with a plurality of samples in, for example, multiplex analysis.
  • the first reporter tag and the second reporter tag may include a different distribution of heavy isotopes.
  • Table I a plurality of peptide reporter tags are shown where G refers to 13 C 2 H 3 15 NO (isotopically heavy glycine) and G refers to C 2 H 3 NO (glycine).
  • the G molecule is 3 atomic mass units (amu's) larger than the normal glycine molecule.
  • Z-B represents a scissile bond
  • groups Z and B may be any of the groups discussed above.
  • B may be proline and Z may be selected from the group consisting of alanine, glutamic acid (or glutamate), phenylalanine, isoleucine, serine, aspartic acid, and asparagine.
  • Z may be aspartic acid (or aspartate) and B may be proline.
  • Z may be asparagine and B is selected from the group consisting of leucine, threonine, and serine.
  • Z and B may represent nucleosides to provide a hybrid nucleic acid-peptide reporter tag.
  • any two or more labels from Table I, or labels similar to those listed in Table I, may be selected as a set and used to analyze a sample.
  • two or more reporter tags are selected such that a fragment from each reporter tag is separated by at least 3 amu's, more particularly by at least about 6 amu's, e.g., fragments are separated by at least 12 amu's.
  • reporter tags having SEQ. ID NO.'s 1 and 4 may be used as a set.
  • reporter tags having SEQ. ID NO.'s 1 and 7 may be used as a set.
  • 12 amu separation is desired, the reporter tags having SEQ.
  • ID NO.'s 1 and 13 may be used as a set.
  • the reporter tags shown in Table I may include a linking group to facilitate attachment of the reporter to a target molecule of interest.
  • the set of reporter tags may further comprise at least one additional reporter tag including at least two scissile bonds.
  • the SUBSTITUTE SHEET at least one additional reporter tag may be constructed and arranged to be distinguished from the first reporter tag and the second reporter tag after cleavage of each of the first reporter tag, the second reporter tag, and the additional reporter tag at one of the at least two scissile bonds.
  • the set of reporter tags comprises at least six different reporter tags, at least ten different reporter tags or at least fifteen different reporter tags.
  • the reporter tags may be selected such that at least 3 amu's separates each reporter tag of the set.
  • a spacing between the first and second scissile bonds of the first reporter tag is different than a spacing between the first and second scissile bonds of the second reporter tag to distinguish the first and second reporter tags from each other.
  • a set of reporter tags 300 is shown.
  • the first reporter tag has a sequence of DPGGGGGGDPGGGGGG (SEQ. ID NO. 88) and the second reporter tag has a sequence of DPGGGGGGGDPGGGGG (SEQ. ID NO. 89).
  • the scissile bonds of the first reporter tag are separated by six glycine residues and the scissile bonds of the second reporter tag are separated by seven glycine residues.
  • Cleavage at one or more of the scissile bonds of the set 300 provides fragment sets 310, 320, 330 and 340. At least one fragment of the fragment sets 310, 320 and 330 may be distinguished from each other on the basis of different mass-to-charge ratios, whereas cleavage of each reporter tag to provide fragment set 340 does not provide distinguishable fragments.
  • the exact spacing between scissile bonds of a reporter tag may vary, and in the case of peptide reporter tags, the spacing between scissile bonds of reporter tags in a set may vary from about six to about ten residues provided that the spacing is different in different reporter tags.
  • the set may further comprise a third reporter tag.
  • the third reporter tag comprises a first scissile bond and a second scissile bond having a spacing from the first scissile bond.
  • the spacing between the second scissile bond and the first scissile bond of the third reporter tag may be selected to distinguish the first, second and third reporter tags from each other.
  • the spacing between the first and second scissile bonds of the first reporter tag is six amino acid residues
  • the spacing between the first and second scissile bonds of the second reporter tag is seven amino acid residues
  • the spacing between the first and second scissile bonds of the third reporter tag is eight amino acid residues.
  • a set of peptide reporter tags comprises a first peptide reporter tag and a second peptide reporter tag is provided.
  • the first peptide reporter tag comprises at least two scissile bonds separated by a first quantity of
  • the second peptide reporter tag comprises at least two scissile bonds separated by a second quantity of amino acids.
  • the first and second quantity of amino acids are different to distinguish the first peptide tag and the second peptide tag.
  • each of the first quantity of amino acids and the second quantity of amino acids is between six and ten provided that the first quantity of amino acids is different than the second quantity of amino acids.
  • the first and second peptide reporter tags are distinguishable after fragmentation in a mass spectrometer. Illustrative peptide reporter tags are shown in Table II below where G represents isotopically heavy glycine ( 13 C 2 H 3 15 NO) and G represents glycine (C 2 H 3 NO).
  • any two or more labels from Table II, or labels similar to those listed in Table II, may be selected as a set and used to analyze a sample.
  • two or more peptide reporter tags are selected such that a fragment from each reporter tag is separated by at least 3 amu's, more particularly by at least about 6 amu's, e.g., fragments are separated by at least 12 amu's.
  • reporter tags having SEQ. ID NO.'s 90 and 93 may be used as a set. If 6 amu separation is desired, then reporter tags having SEQ. ID NO.'s 90 and 96 may be used as a set. If 12 amu separation is desired, the reporter tags having SEQ. ID NO.'s 90 and 102 may be used as a set.
  • the peptide reporter tags shown in Table II may include a linking group to facilitate attachment of the reporter to a target molecule of interest.
  • a kit comprising at least two reporter tags is disclosed.
  • the kit comprises a first reporter tag including at least two scissile bonds.
  • the kit further comprises a second reporter tag including at least two scissile bonds.
  • the first reporter tag is constructed and arranged to be distinguished from the second reporter tag after cleavage of each of the first reporter tag and the second reporter tag at one of the at least two scissile bonds.
  • the kit further comprises instructions for using the first and second reporter tags.
  • each of the first reporter tag and the second reporter tag may be configured as a peptide reporter tag.
  • the kit may further comprise at least one additional reporter tag including at least two scissile bonds.
  • the at least one additional reporter tag may be constructed and arranged to be distinguished from the first reporter tag and the second reporter tag after cleavage of each of the first reporter tag, the second reporter tag, and the additional reporter tag at one of the at least two scissile bonds.
  • a method comprising separating a first doubly- scissiled reporter tag coupled to a first target molecule and a second doubly- scissiled reporter tag coupled to a second target molecule.
  • a double- scissiled reporter tag refers to a reporter tag having two scissile bonds, as discussed herein.
  • Illustrative separation methods are discussed herein and include, but are not limited to, electrophoresis, chromatography and mass spectrometry. Additional methods for separating doubly-scissiled reporter tags will be readily selected by the person of ordinary skill in the art, given the benefit of this disclosure.
  • the method may further comprise cleaving the first and second doubly-scissiled reporter tags.
  • Cleavage may be accomplished by numerous methods including chemical cleavage, enzymatic cleavage or physical cleavage using, for example, collisional dissociation.
  • the person of ordinary skill in the art, given the benefit of this disclosure, will be able to select additional methods for cleaving doubly-scissiled reporter tags.
  • the method may further comprise distinguishing the first doubly- scissiled reporter tag and the second doubly-scissiled reporter tag.
  • the reporter tags may be distinguished by detection of a high mass fragment, a low mass fragment, or both, using mass spectrometry.
  • the method may further comprise distinguishing the first doubly-scissiled reporter tag and the second doubly-scissiled reporter tag by fragmenting the first doubly-scissiled reporter tag and the second doubly-scissiled reporter tag in a mass spectrometer and detecting at least one fragment from one of the fragmented, first doubly-scissiled reporter tag and the fragmented, second doubly-scissiled reporter tag.
  • the method may further comprise detecting at least one fragment from each of the fragmented first doubly-scissiled reporter tag and the fragmented second doubly-scissiled reporter tag.
  • the reporter tags may be distinguished by separating the reporter tags and detection using an immunoassay or other analytical techniques.
  • the method may further comprise configuring
  • the method may further comprise determining an amount of each of the first target molecule and the second target molecule. In other examples, the method may further comprise comparing the amount of the first target molecule and the amount of the second target molecule. In some examples, the first and second target molecules may be the same molecule but from a different source, e.g., insulin from a first patient and insulin from a second patient.
  • the method comprises providing a set comprising a first reporter tag including at least two scissile bonds and a second reporter tag including at least two scissile bonds, the first reporter tag constructed and arranged to be distinguished from the second reporter tag after cleavage of each of the first reporter tag and the second reporter tag at one of the at least two scissile bonds.
  • the method further comprises providing instructions for using the set to sequence a peptide.
  • a mass spectrometer may comprise a first stage configured to select at least two target molecules labeled with two, different reporter tags.
  • the mass spectrometer may also comprises a second stage in fluid communication with the first stage, the second stage configured to fragment the two, reporter tags of the selected at least two target molecules to provide at least two low mass fragments and two high mass fragments.
  • fragmentation may produce three fragments for each of the reporter tags.
  • the mass spectrometer may further include a processing device electrically coupled to the second stage, the processing device configured to quantify simultaneously the two or more target molecules.
  • the first stage and the second stage may each be configured as a quadrupole mass spectrometer.
  • the first stage may be configured to pass two or more target molecules with a mass-to-charge ratio within a selected mass-to-charge window.
  • the processing device may include an algorithm that is configured to quantify the target molecules by fitting a mathematical function to the two low mass fragments to quantify simultaneously a relative amount of the at least two target molecules. Illustrative algorithms are discussed in the commonly assigned patent application (U.S. 11/464,930) incorporated by reference above.
  • fractionation systems integrated with mass spectrometers are commercially available, and illustrative systems include liquid chromatography (LC) and capillary electrophoresis (CE).
  • the components of a mass spectrometer typically include: (a) one or more sources, (b) one or more analyzers and/or cells, and (c) one or more detectors.
  • Types of sources include, but are not limited to, Electrospray Ionization (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI).
  • Types of analyzers and cells include, but are not limited to, quadrupole mass filter, hexapole collision cell, ion cyclotron trap, and Time-of- Flight (TOF).
  • Types of detectors include, but are not limited to, Multichannel Plates (MCP) and ion multipliers.
  • tandem mass spectrometers with more than one analyzer/cell are known as tandem mass spectrometers.
  • tandem mass spectrometers There are two types of tandem mass spectrometers, as well as hybrids and combinations of these types: "tandem in space” spectrometers and "tandem in time” spectrometers.
  • Tandem mass spectrometers where the ions traverse more than one analyzer/cell are known as tandem in space mass spectrometers. Tandem in space spectrometers use spatially ordered elements and act upon the ions in turn as the ions pass through each element.
  • Tandem mass spectrometers where the ions remain primarily in one analyzer/cell are known as tandem in time mass spectrometers.
  • Tandem in time spectrometers utilize temporally ordered manipulations on the ions as the ions are contained in a space. Hybrid systems and combinations of these types are known.
  • the ability to select a particular mass-to-charge ratio of interest in a mass analyzer is typically characterized by the resolution (reported as the centroid mass-to-charge divided by the full width at half maximum of the selected ions of interest).
  • resolution is an indicator of the narrowness of the ion mass-to- charge distribution passed through the analyzer to the detector. Reference to such resolution is generally noted herein by referring to the ability of a mass spectrometer to pass only a narrow range of mass-to-charge ratios.
  • tandem mass spectrometer such as a tandem in space tandem mass spectrometer.
  • the reporter tags may be first passed through a filtering quadrupole, the reporter tags may be fragmented (preferably in a collision cell), and the fragments may be distinguished and detected in a time-of-flight (TOF) stage.
  • TOF time-of-flight
  • the sample is ionized in the source (for example, in a MALDI ion source) to produce charged ions. It is preferred that the ionization conditions are such that primarily a singly charged parent ion is produced.
  • a first quadrupole, QO is operated in radio frequency (RF) mode only and acts as an ion guide for all charged particles.
  • the second quadrupole, Ql is operated in RF+DC mode to pass only a narrow range of mass-to-charge ratios (that includes the mass-to-charge ratio of the reporter signals). This quadrupole selects the mass-to-charge ratio of interest.
  • Quadrupole Q2, surrounded by a collision cell is operated in RF only mode and acts as ion guide. The collision cell
  • SUBSTITUTE SHEET surrounding Q2 may be filled to appropriate pressure with a gas to fracture the input ions by collisionally induced dissociation when fragmentation of the reporter signals is desired.
  • the collision gas preferably is chemically inert, but reactive gases may also be used.
  • Preferred molecular systems utilize reporter tags that contain scissile bonds, labile bonds, or combinations, such that these bonds will be preferentially fractured in the Q2 collision cell. [0059] Tandem instruments capable of MS n can be used with the disclosed method.
  • Example 1 A set of reporter signals having the following sequences was used.
  • Tag 1 Iodine- Ac-DPGGGGGGDPGGGGGGGG (SEQ. ID NO. 177)
  • Tag 2 Iodine-Ac-DPGGGGGGGDPGGGGG (SEQ. ID NO. 178)
  • Tag 3 Iodine-Ac-DPGGGGGGDPGGGG (SEQ. ID NO. 179)
  • a target peptide having the sequence Gly-Tyr-Ser-Leu-Gly-Asn-Trp-Val-Cys-Ala- AIa- Lys (SEQ ID NO. 180) was analyzed. Three groups of target peptides each containing identical amounts of the target peptide were reacted under suitable conditions with Tags 1, 2 and 3 listed above. The iodoacetyl group on the N-terminus of each of the tags will form a covalent bond with a sulfhydryl group on the cysteine residue in the target peptide at physiological pH (e.g., pH of 7.0), thus freeing the iodine. The product of the reporter tag and peptide reaction is shown below.
  • the three reporter tag-coupled target peptides were then pooled and analyzed by tandem mass spectrometry. Because all of the reporter tags have the same mass (and therefore the same mass:charge ratio (i.e., m/z)), the three groups of reporter tag-coupled target peptides each had the same predicted mass-to-charge, and the same atomic mass unit of the reporter tag-coupled target peptides.
  • MSl mass:charge ratio
  • the target proteins in the selected peak were subjected to collision induced dissociation (CID).
  • CID collision induced dissociation
  • the two scissile bonds in each of the three tags broke between the aspartic acid (D) and the proline (P) residues.
  • the fragments released by the broken reporter tag-coupled target peptides will depend upon which of the three tags was coupled to the target peptide.
  • all three reporter tag-coupled target peptides when the N-terminal DP scissile bond is broken, resulted in a fragment peaking at a mass-to-charge of about 1423.7846.
  • the relevant peaks in FIG. 4 from the lowest amu (or m/z ratio) to the highest, observed are listed in Table III below.
  • Example 2 A set of 18 reporter tags comprising SEQ. ID NO.'s 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49 and 52 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.
  • Example 3 A set of 15 reporter tags comprising SEQ. ID NO.'s 4, 7, 10, 13, 19, 22, 25, 28, 31, 37, 40, 43, 46, 49 and 52 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.
  • Example 4 A set of 10 reporter tags comprising SEQ. ID NO.'s 1, 7, 13, 16, 22, 28, 34, 40, 46 and 52 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.
  • Example 5 A set of 7 reporter tags comprising SEQ. ID NO.'s 34, 37, 40, 43, 46, 49 and 52 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.
  • SUBSTITUTE SHEET [0069] A set of 4 reporter tags comprising SEQ. ID NO.'s 34, 40, 46 and 52 is provided. The set of reporter tags may be used in many different analyses including multiplex analysis.
  • Example 7 A set of 2 reporter tags comprising SEQ. ID NO.'s 37 and 49 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.
  • a set of 18 reporter tags comprising SEQ. ID NO.'s 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, and 141 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.
  • a set of 15 reporter tags comprising SEQ. ID NO.'s 93, 96, 99, 102, 108, 111, 114, 117, 120, 126, 129, 132, 135, 138 and 141 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.
  • a set of 10 reporter tags comprising SEQ. ID NO.'s 90, 96, 102, 105, 111, 117, 123, 129, 135 and 141 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.
  • a set of 7 reporter tags comprising SEQ. ID NO.'s 123, 126, 129, 132, 135, 138 and 141 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.
  • a set of 4 reporter tags comprising SEQ. ID NO.'s 123, 129, 135, and 141 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.
  • a set of 2 reporter tags comprising SEQ. ID NO.'s 126 and 138 is provided.
  • the set of reporter tags may be used in many different analyses including multiplex analysis.

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Abstract

La présente invention concerne un groupe constitué d'un premier marqueur rapporteur incluant au moins deux liaisons susceptibles d'être scindées et un second marqueur rapporteur incluant au moins deux liaisons susceptibles d'être scindées. Selon certains exemples de l'invention, le premier marqueur rapporteur peut être élaboré et adapté de façon à le distinguer du second marqueur rapporteur après le clivage de chacun des premier marqueur rapporteur et second marqueur rapporteur au niveau d'une ou plusieurs des deux liaisons au moins susceptibles d'être scindées. L'invention porte également sur des procédés d'utilisation des marqueurs rapporteurs.
PCT/US2008/051773 2007-01-24 2008-01-23 Marqueurs rapporteurs pouvant être scindés et procédés d'utilisation Ceased WO2008091930A2 (fr)

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